Targeted cancer therapies often induce “outlier” responses in molecularly defined patient subsets. One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a near-complete clinical and radiographic remission for 5 years. Whole-genome sequencing and RNA sequencing of primary tumor and normal samples from this patient identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutations were shown to transform immortalized human airway epithelial cells in a sorafenib-sensitive manner. These results suggest that mutant ARAF is an oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.

Lung adenocarcinomas harbor recurrent activating oncogenic mutations and fusions in receptor tyrosine kinase pathway genes, some of which (EGFR, EML4-ALK, CD74-ROS1) have been associated with clinical response to small-molecule inhibition (1–3). Despite these advances, most lung adenocarcinoma cases lack a clinically actionable genetic alteration and over 50% lack a plausible oncogenic “driver,” as demonstrated by recent large-scale genome surveys (3). One approach for the discovery of clinically actionable drivers is genomic analysis of exceptional drug responses (4). We used next-generation sequencing to investigate the genetic basis of a sustained “outlier” response to sorafenib in lung adenocarcinoma.

A 66-year-old light former smoker (<5 packs per year smoking history) was diagnosed in April 2002 with stage IV lung adenocarcinoma. She failed multiple therapy regimens (gemcitabine and vinorelbine, gefitinib, bortezomib) between 2002 and 2005 and received a palliative lobectomy in early 2006 for worsening hypoxia. She began treatment with oral sorafenib, a broad-spectrum kinase inhibitor with activity against BRAF, RAF1, RET, PDGFRA, and KIT, among others (5, 6), in June 2006 as part of the ECOG 2501 trial (7). Within 2 months, her CT scans demonstrated a near-complete response (Figure 1). She remained progression free and asymptomatic for the next 5 years while continuing sorafenib treatment. In July 2011, a CT scan demonstrated enlargement of a right lower lobe mass meeting Response Evaluation Criteria In Solid Tumors criteria for progression. Sorafenib was discontinued, and she was started on carboplatin, paclitaxel, and bevacizumab. Therapy was discontinued after 2 cycles due to side effects, worsening fatigue, and oxygen requirements. She was admitted to hospice and died in November 2011. At the time of relapse, she was the last remaining study participant receiving sorafenib and 1 of only 9 responders among 306 evaluable patients. A more detailed time line of her case is shown in Figure 1 and described in the Supplemental Methods (supplemental material available online with this article; doi:
10.1172/JCI72763DS1).

Time line of patient’s lung adenocarcinoma diagnosis, treatment, and response. Colored rectangles near the time line represent durations of targeted therapy (blue, green) and chemotherapy (pink). Original magnification, ×200 (Apr. 2002). Dx, diagnosis; RLL, right left lobe; RML, right middle lobe.

Among 101 somatic coding mutations and 2 in-frame fusions predicted by WGS analysis, only 15 variants were detected in RNA sequencing (RNA-seq) data with more than 2 supporting reads (Table 1). Among expressed coding variants, the most likely candidate oncogenic driver was ARAF S214C. ARAF encodes a serine-threonine kinase in the Raf protein family, which also includes RAF1 (also known as CRAF) and BRAF, a lung adenocarcinoma and melanoma oncogene. Like other Raf family proteins, ARAF transduces MAP kinase pathway signals from Ras to MEK and ERK and is a sorafenib target; however, unlike its paralogs, ARAF has never been implicated in tumorigenesis (6, 13).

In summary, our genomic and functional results suggest the transforming ARAF codon 214 substitution as the most likely driver of this patient’s tumor and determinant of its sorafenib response. The absence of known recurrent oncogenic alterations or alterations in other sorafenib targets (Supplemental Table 4) in our comprehensive profiling data supports this conclusion. Moreover, the discovery of additional patients with transforming ARAF codon 214 and RAF1 codon 257 and 259 mutations in independent lung adenocarcinoma and pan-cancer data sets suggests somatic selection for a novel oncogenic hot spot in ARAF and RAF1, both which encode sorafenib targets (6). Inhibition of colony formation by sorafenib or trametinib in cells overexpressing wild-type ARAF or RAF1 suggests that sorafenib and trametinib do not exhibit increased activity toward the mutant gene products, but rather that ARAF and RAF1 mutations may confer an inhibitor-sensitive oncogene dependency. This is consistent with the current understanding of the mechanisms underlying the specificity of established targeted therapies (e.g., imatinib) (17). To the best of our knowledge, the ARAF and RAF1 mutations profiled in this study have not been characterized previously as oncogenic somatic mutation hot spots in clinical cancer samples. Interestingly, one group has recently associated derived RAF1 p.S257P mutations with in vitro PLX-4720 resistance in BRAF p.V600E mutant melanoma cells (18); however, all of the TCGA samples from patients with lung cancer analyzed in the current study were treatment naive.

The ARAF/RAF1 mutations characterized in this study lie in a Raf CR2-domain phosphorylation site that negatively regulates Ras binding and RAF1 activation via binding of 14-3-3 (13, 19). This region is distinct from the kinase domain hot spots of BRAF (near p.G469 and p.V600), which are mutated in approximately 5% of patients with lung adenocarcinoma (3), suggesting that ARAF p.S214/RAF1 p.S259 may function by a distinct biochemical mechanism. Our preliminary biochemical data suggests that ARAF also bound 14-3-3, and this binding was markedly attenuated in the setting of p.S214C mutation (Supplemental Figure 7). These results suggest that ARAF p.S214 mutations may potentiate Ras/Raf signaling by abrogating 14-3-3 and ARAF binding.

Clinical trials of sorafenib in patients with advanced non–small-cell lung cancer have demonstrated modest activity, with no survival advantage (7, 20). Though it is attractive to consider ARAF/RAF1 mutations as possible biomarkers of sorafenib response in lung adenocarcinoma, additional profiling and functional characterization studies will be required to establish this link. This includes mutation data from additional sorafenib responders and/or sorafenib-response data from ARAF p.S214/RAF1 p.S259 mutated cases. Our initial sequencing of 3 transient responders (<6-month progression-free survival) from the ECOG 2501 trial did not reveal additional Raf family mutations. Identification of additional ARAF p.S214/RAF1 p.S257 mutant cases in lung adenocarcinoma will likely require screening of large sets of patients, given the low frequency of these variants (1%). Though we believe that the cell line model used in this study should adequately recapitulate the observed overexpression of mutant ARAF in this patient’s tumor (Table 1 and Supplemental Table 4), knockdown and inhibitor studies of cell lines with naturally occurring ARAF/RAF1 mutations will be useful in definitively establishing essentiality and oncogene addiction in an endogenous context. Our preliminary analysis of the Cancer Cell Line Encyclopedia sequencing data (
http://www.broadinstitute.org/ccle/home) did not reveal any ARAF p.S214/RAF1 p.S257 mutant cell lines.

If recurrent but rare mutations underlie the oncogenicity and responsiveness of “driver-negative” lung adenocarcinomas, they are not likely to be nominated by statistical analysis of several hundred (or even thousands) of genome-sequenced cases. Our study suggests that a powerful, alternate approach to driver mutation discovery may be through the analysis of outlier patient responses and the identification of driver mutations through the preponderance of genomic, biochemical, and functional evidence.

Retroviral transduction and soft agar assays. Ectopic expression of mutant constructs and assessment of anchorage-independent proliferation were performed as described previously (21). See the Supplemental Methods for experimental details.

Study approval. The collection and genomic analysis of this patient sample was carried out in accordance with protocols approved by Institutional Review Boards at the Broad Institute of Harvard and MIT (Broad COUHES 1103004402) and Vanderbilt University (VICC THO 0547). Informed consent for genomic analysis was obtained from the patient at the time of sample collection.

M. Imielinski is supported by NCI training grant T32 CA9216-31. L. Araujo is supported by a Conquer Cancer Foundation of ASCO Long-Term International Fellowship. This work was supported by NCI U01CA114771 (to D.P. Carbone), Uniting Against Lung Cancer (to M. Meyerson and H. Greulich), the Lung Cancer Research Foundation (to M. Meyerson), Novartis (to M. Meyerson), Department of Defense Congressionally Directed Medical Research Programs Lung Cancer Research Program W81XWH-12-1-0269 (to M. Meyerson), and the American Lung Association (to M. Meyerson). We thank Mark Bray and members of the Broad Institute Imaging Platform for development of the CellProfiler pipeline, used to quantify the soft agar photographs.